Multiplex FRET Imaging of Kinase-Epigenome Interregulations in Live Cancer Cells Kinase inhibitors have been applied to mitigate pancreatic cancer development. However, adaptive epigenetic responses including histone modulations can lead to the alteration of large scale gene expressions which can ultimately result in heterogeneous drug resistant responses of cancer cells and life-threatening relapse of diseases. At the current stage, it remains unclear how tyrosine kinase activities are dynamically coupled with epigenetic histone modulations to determine cancer cell responses upon drug treatment. Therefore, investigating and manipulating the regulation of histone modulations and codes have crucial implications in cancer treatment and drug screening. In this proposal, we will harness the power of directed evolution and high-content screening methods to systematically develop fluorescence resonance energy transfer (FRET) biosensors for the dynamic monitoring and quantification of crucial histone methylations (H3K4, H3K9, H3K27) in single cells. We will also apply this approach to optimize a focal adhesion kinase (FAK) FRET biosensor for the visualization of FAK kinase activity. Together with an existing Src FRET biosensor optimized by us, these biosensors will be incorporated into the genome of pancreatic cancer cells using CRIPSR to minimize the heterogeneity of signals across different individual cells. We will further incorporate new FRET pairs emitting colors distinct from the popular FRET pair (CFP and YFP) to simultaneously monitor two different signals in the same live cell, e.g. one histone methylation and one kinase activation. Using a common molecular signal as a reference across different individual cells, these crucial molecular events will be mapped together with correlative FRET imaging method (CFIM) developed in our labs to generate dynamic landscapes of kinome-epigenome interactions. Pharmacological reagents will be applied to study their impact on these dynamic landscape of molecular interactions and adaptive epigenetic responses. We will then correlate these multiplex molecular profiles to cancer outcomes under these pharmacological reagents, and hence provide quantified multiplex indices to evaluate drug efficacy at the single-cell level with the goal of minimizing drug resistance. Three specific aims are accordingly proposed: (1) Develop and optimize histone methylation and tyrosine kinase FRET biosensors; (2) Apply CRISPR to genetically engineer FRET biosensors into pancreatic cancer cell lines for the calibration of inhibitor efficacy in single cells; (3) Multiplex imaging of histone methylations and kinase activities in the same PDAC cells for assessing adaptive epigenetic responses upon kinase inhibition. Given the importance and critical needs of new imaging tools to investigate the kinome-epigenome connection in cancer cells, developed FRET biosensors and imaging system should provide powerful means to unravel the molecular network for cancer biology, and allow multiplex and high throughput platform for drug screening with minimal resistance. As such, the success of the project will contribute transformative enabling technologies to the field of cancer research and pharmaceutics, toward an ultimate goal of eradicating pancreatic cancers.